CN112695021A - 一种α-糖苷酶基因突变体及在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 - Google Patents
一种α-糖苷酶基因突变体及在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 Download PDFInfo
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Abstract
本发明公开了一种α‑糖苷酶基因突变体及在制备2‑O‑α‑D‑葡萄糖基‑L‑抗坏血酸中的应用，所述α‑糖苷酶基因突变体是将第270位酪氨酸突变为苯丙氨酸，或/和将第373位色氨酸分别突变为亮氨酸，或/和将第411位甲硫氨酸突变为苯丙氨酸，或/和将第491位精氨酸突变为赖氨酸，或/和将第504位色氨酸突变为亮氨酸所得。该突变体制备简单，实现了催化生成AA‑2G的产量的提高。相同条件下，突变体比活力较原始酶明显提高，催化生成AA‑2G的产量最高可达18.9 g/L，产AA‑2G的转化率较原始酶有显著的提高。
Description
技术领域
本发明属于基因工程和酶工程领域，特别涉及一种来源于Oryza sativa的α-葡萄糖苷酶突变体在制备2-O-α-D-葡萄糖基-L-抗坏血酸（AA-2G）中的应用。
背景技术
L-抗坏血酸(又称维生素C，VC)是一种人体自身不能合成的水溶性维生素，参与体内多种生理活动个，如促进胆固醇转变为胆汁酸，促进肾上腺皮质激素的合成，参与芳香族氨基酸的代谢，促进铁的吸收及参与体内多种氧化还原反应等，在维持和促进人体健康中扮演着重要的角色。然而其C-2位上的羟基极不稳定，在氧、热、光、重金属等条件下非常容易发生氧化降解，导致其生理活性减弱甚至消失，从而极大的限制了其应用。
上个世纪以来，开发既保证VC正常生理功能又稳定的VC衍生物成为国内外学者研究的热点，目前主要的VC衍生物有金属盐类、酯类及糖基类衍生物。与VC其他的衍生物相比，2-O-α-D-葡萄糖基-L-抗坏血酸（AA-2G）稳定性强，安全性高，生产方法简单，进入机体后，可以在α-葡萄糖苷酶的作用下缓慢分解成VC与D-葡萄糖，这种缓释作用能长效保持VC在体内发挥正常的功能，是最佳的VC替代品，目前被广泛应用于食品、化妆品、医疗保健等行业。
利用糖基转移酶的特异性转糖基作用进行生物转化合成是目前维生素C葡萄糖苷的唯一生产途径。目前所报道的AA-2G生物催化用酶主要有α-葡萄糖苷酶（EC 3 .2 .1.20，α-Glucosidase）、环麦芽糊精葡聚糖转移酶（EC 2 .4 .1 .19，CyclomaltodextrinGlucanotransferase，简称CGTase）、α-淀粉酶（EC 3 .2 .1 .1，α-Amylase）、蔗糖磷酸化酶（EC 2 .4 .1 .7，Sucrose phosphorylas）、葡聚糖蔗糖酶（EC 2 .4 .1 .5，Dextransucrase）、α-异麦芽糖基葡糖基形成酶等六类酶。1990年，NorioMuto等首次发现大米来源的α-葡萄糖苷酶能够以成本较低的麦芽糖为糖基供体，特异性催化L-AA合成AA-2G。但相关研究较少。
发明内容
本发明的目的是提供一种利用α-葡萄糖苷酶突变体制备2-O-α-D-葡萄糖基-L-抗坏血酸（AA-2G）的方法，实现生物法一步催化转化高效生产AA-2G，具有产物单一易于分离纯化的特点。
本发明采用的技术方案是：
一种α-葡萄糖苷酶突变体在制备2-O-α-D-葡萄糖基-L-抗坏血酸（AA-2G）中的应用，所述α-葡萄糖苷酶突变体以来源于Oryza sativa（UniProtKB登录号：Q653V7）的α-葡萄糖苷酶（其氨基酸序列如SEQIDNO.1所示）为亲本，并对其进行一个或多个氨基酸的突变所得。
本发明所述的α-葡萄糖苷酶突变体包括与SEQ ID NO.1所示氨基酸序列≥95％同源性(优选地，≥96％的同源性；更优选地≥97％的同源性；最优选地，≥98％的同源性，如≥99％的同源性)的多肽，且所述多肽具有催化活性；以及将SEQ ID NO .1中所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的多肽。
一种重组表达载体，它包含所述的α-葡萄糖苷酶突变体基因。可以使用本领域公知的方法将重组载体构建为克隆或表达用载体。任何适合在基因重组中使用的载体都可以用作重组载体。详细地，重组载体可以适于在毕赤酵母中表达，并且可以基于选自由pAO、pGAP、pPIC和pPINK组成的组中的一种进行构建。
只要具有允许蛋白表达（过量表达）的表达系统，任何微生物都可以用作重组载体转化为的宿主细胞。例如，毕赤酵母可能是有用的。毕赤酵母的实例包括但不限于：GS115、X-33、KM71、KM71H、SMD1168和SMD1168H。此外，宿主细胞还可以是大肠杆菌，如 BL21、JM109等。
一种重组菌，其可以是通过所述的表达载体转化宿主细胞获得，也可以通过将所需表达的多核苷酸序列转入宿主细胞基因组中获得。
本发明提出一种用于生产AA-2G的组合物，包含选自由以下组成的组中的一种及以上：包含SEQIDNO.1的氨基酸序列或基本由该氨基酸序列组成的蛋白、编码该蛋白的多核苷酸、携带该多核苷酸的重组载体、含有该重组载体的重组细胞、该重组细胞的培养物、该重组细胞的培养物上清液和该重组细胞的裂解物。
在本发明的一种实施方式中，所述突变体相对于亲本，将亲本第270位酪氨酸突变为苯丙氨酸，简称Y270F，其多核苷酸序列如SEQIDNO.3所示。
在本发明的一种实施方式中，所述突变体相对于亲本，将亲本第373位色氨酸突变为亮氨酸，简称W373L，其多核苷酸序列如SEQIDNO.4所示。
在本发明的一种实施方式中，所述突变体相对于亲本，将亲本第411位甲硫氨酸突变为苯丙氨酸，简称M411F，其多核苷酸序列如SEQIDNO.5所示。
在本发明的一种实施方式中，所述突变体相对于亲本，将亲本第491位精氨酸突变为赖氨酸，简称R491K，其多核苷酸序列如SEQIDNO.6所示。
在本发明的一种实施方式中，所述突变体相对于亲本，将亲本第504位色氨酸突变为亮氨酸，简称W504L，其多核苷酸序列如SEQIDNO.7所示。
该突变体制备简单，实现了酶活力显著提高且催化生成AA-2G的产量的提高。相同条件下，突变体比活力较原始酶明显提高，最高可达10.82 U/mg，提高约12.4倍；催化生成AA-2G的产量最高可达18.9 g/L，提高约15.75倍，产AA-2G的转化率较原始酶有显著的提高，产物单一，易于分离纯化。
以包含但不限于麦芽糖、麦芽低聚糖、异麦芽糖、蔗糖、曲二糖和淀粉为糖基供体、L-抗坏血酸为糖基受体，转化生成2-O-α-D-葡萄糖基-L-抗坏血酸。
所述糖基供体包含但不限于100~300g/L的麦芽糖、100~300g/L的麦芽低聚糖、100~300g/L的异麦芽糖、100~300g/L的蔗糖、100~300g/L的曲二糖和20~100g/L淀粉。
所述糖基受体为30-50g/L L-抗坏血酸，催化剂浓度为10-20 U/mL。
所述反应在30℃-55℃、pH4.0-5.0条件下反应1-6h。
本发明提出一种生产AA-2G的方法，其最佳反应条件为以100 g/L的麦芽糖为糖基供体，35 g/L L-抗坏血酸为糖基受体，10-20 U/mLα-葡萄糖苷酶突变体W373L作为催化剂，于37℃、pH4.0条件反应6h生成的AA-2G的产量达到最高，为18.9 g/L。
有益效果：本发明基于生物信息学的分析方法和分子生物学技术对来源于Oryza sativa的α-葡萄糖苷酶进行改造，得到α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L。所得α-葡萄糖苷酶突变体较本体，酶活力有明显提高，且大幅提高了生物合成AA-2G应用中的转化率及产物量。因此，预期根据本发明的利用该酶生产AA-2G的方法可以为酶法生成AA-2G提供新思路。
附图说明
图1是根据本发明的一种实施方式的用于表达α-葡萄糖苷酶蛋白的重组载体的图谱；
图2是α-葡萄糖苷酶分泌表达及纯化后的SDS-PAGE电泳图，M，Takara SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）中分子量标准蛋白；1：摇瓶发酵后分泌表达的重组α-葡萄糖苷酶；2：经蛋白纯化后的α-葡萄糖苷酶。箭头标注为目的蛋白。
图3是根据本发明的一种实施方式的利用α-葡萄糖苷酶催化生成AA-2G的HPLC色谱图。
具体实施方式
下面的实例可以使本领域技术人员更全面地理解本发明，但不以任何方式限制本发明。
YPD培养基：酵母提取物10g·L-1，胰蛋白胨20g·L-1，葡萄糖20g·L-1。
BMGY培养基：酵母提取物10g·L-1，胰蛋白胨20g·L-1，YNB 13.4g·L-1，高压灭菌后加入2mL·L-1 0.02%生物素，100mL·L-1 1M磷酸钾缓冲溶液（pH 6.0），100mL·L-1 10%甘油。
BSM培养基：40g·L-1甘油，26.7mL·L-1 85%H3PO4，0.93g·L-1CaSO4，18.2g· L-1K2SO4，14.9g· L-1 MgSO4·7H2O，4.13g·L-1 KOH，酵母提取物10g·L-1，胰蛋白胨20g·L-1。
α-葡萄糖苷酶酶活测定：在100 μl反应体系中（25 mM麦芽糖，100 mM乙酸缓冲，pH5.0）加入15 μg粗酶液启动反应，反应在37℃水浴锅条件下进行，10 min后反应液经95℃水浴加热7 min后终止反应。后利用葡萄糖氧化酶法检测样品中生成的葡萄糖的量。
α-葡萄糖苷酶的酶活定义为：在上述条件下，每分钟水解1 μmol底物所需的酶量称为一个酶活力单位（U）。
AA-2G的高效液相色谱（HPLC）检测：色谱柱为Agilent 5 TC-C18柱，5 μm（φ250×4 .6 mm）。紫外检测波长为 238 nm。流动相为 1 %的甲醇，用磷酸调pH至 2 .0，流速 0 .8mL/min，时间为10 min，进样量10 μL，柱温为 25 ℃。HPLC图谱中AA-2G的峰面积和浓度成正比，以此绘制AA-2G标准曲线。利用标准曲线及样品中的AA-2G的峰面积，得出样品中AA-2G的浓度。
实施例1：重组载体（pGAP9K-AGL）的构建
来源于Oryza sativa subsp. japonica (Rice)的α-葡萄糖苷酶基因，其核苷酸序列如SEQ ID NO.2所示，由南京金斯瑞科技有限公司（GenScript）合成该基因后克隆到真核表达载体pGAP9K的限制性内切核酸酶位点EcoRI后，且使其N-末端添加6-his标签，得到重组载体pGAP9K-AGL（参考图1）。
实施例2：α-葡萄糖苷酶突变体的制备
根据如序列SEQ NO.2所示的α-糖苷酶的核苷酸序列，设计并合成引入有Y270F、W373L、M411F、R491K和W504L突变的引物，利用定点突变试剂盒（Vazyme. Co.）以实施例1所得重组质粒pGAP9K-AGL为模板，经PCR扩增、产物DpnI消化、重组转化、质粒抽提、测序验证等步骤得到在相应位点进行突变的突变体重组质粒pGAP9K-AGL-Y270F、pGAP9K-AGL-W373L、pGAP9K-AGL-M411F、pGAP9K-AGL-R491K和pGAP9K-AGL-W504L。
引入Y270F突变的定点突变引物为（下划线为突变碱基）：
正向引物：SEQID NO.8 5’ TGTAGATTCGGTTATAAGAACGTTGCTGATT 3’
反向引物：SEQID NO.9 5’ ATAACCGAATCTACATTGGTGAAAACCGAA 3’
引入W373L突变的定点突变引物为（下划线为突变碱基）：
正向引物：SEQID NO.10 5’ TGTTGTTTGGCCAGGTAACGTTTACTTCCC 3’
反向引物：SEQID NO.11 5’ ACCTGGCCAAACAACACCCAAGTAGTTAGAA 3’
引入M411F突变的定点突变引物为（下划线为突变碱基）：
正向引物：SEQID NO.12 5’ TGGGTTGATTTCAACGAGATTTCTAACTTTG 3’
反向引物：SEQID NO.13 5’ CTCGTTGAAATCAACCCACAAACCATCAAC 3’
引入R491K突变的定点突变引物为（下划线为突变碱基）：
正向引物：SEQID NO.14 5’ TTTGTCTAAAAGCACTTTCGTTGGTTCTGG 3’
反向引物：SEQID NO.15 5’ AAGTGCTTTTAGACAAAACGAATGGTCTTCT 3’
引入W504L突变的定点突变引物为（下划线为突变碱基）：
正向引物：SEQID NO.16 5’ GCTCATTTGACTGGTGACAACGCTGCTACT 3’
反向引物：SEQID NO.17 5’ ACCAGTCAAATGAGCAGTATATCTACCAGAACC 3’
利用限制性内切酶PshAI（New England Biolabs）将上述所得突变体重组质粒酶切消化并胶回收后得到线性化突变体重组载体，并通过电转化法电转到提前准备好的PichiapastorisGS115感受态细胞中，在30℃下MD平板培养3-4天，长出转化子后挑至不同浓度的G418/YPD平板上进行阳性克隆子的筛选，经30℃培养24-36 h。挑选单克隆进行菌落PCR，验证正确的克隆转接到10 mL的YPD液体培养基中，30℃、200 rpm培养24 h，保存甘油管，贮存于-80℃冰箱；并进行摇瓶发酵产酶。
实施例3：摇瓶发酵产酶
将实施例2中得到的重组毕氏酵母菌株Y270F、W373L、M411F、R491K和W504L分别接种于YPD培养基中，在30℃下培养24h后以10％接种量转接到50mL BMGY培养基中于30℃、200rpm摇床中培养18 h，然后4000 rpm 离心5min，弃上清，收集菌体，用200 mL BMGY培养基垂悬菌体，于30℃恒温摇床中培养72h，每12h补加补充终浓度为1%葡萄糖作为碳源。摇瓶发酵结束后，5000rpm离心20min，上清即为所需α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L的粗酶液。选择蛋白表达量较高且酶活力较高的重组毕赤酵母菌株用于发酵罐连续发酵培养。
实施例4：5L发酵罐发酵产酶
将经实施例3中摇瓶发酵后筛选出的高表达重组毕赤酵母菌株接种于YPD培养基中，在30℃、200 rpm培养24h后得到一级种子液；以10％接种量转接到200 mL BMGY培养基并培养约18h（OD值达到18~20），制备得到二级种子液；以10%接种量转接到含有2LBSM改进培养基（含4.35 ml/L PTM1溶液）的5L发酵罐中，设定发酵初始条件为温度30℃、转速600 r/min、通气比1.5 vvm、pH 5.0开始发酵。发酵约16h左右，初始培养基中的碳源消耗完，溶氧开始反弹，此时开始流加50%甘油（含12ml/L PTM1溶液），流速控制在16 ml/L/h，补料3h后调整补料速度保持为10 ml/L/h，持续补料84h。整个发酵过程中。控制溶氧水平于20%左右。发酵过程中每6h取样，利用紫外分光光度计测定其OD值；6000 rpm离心20min后测定菌体湿重并使用Brandford法测定粗酶液的蛋白浓度。发酵罐发酵结束后，6000 rpm离心20min，上清即为重组α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L的粗酶液。
实施例5：α-葡萄糖苷酶突变体的分离纯化
利用金属亲和层析对经实施例4得到的重组α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L进行纯化。首先，将5 ml Ni -NTA柱安装至蛋白纯化仪，用缓冲液A（50 mM磷酸钠缓冲，300 mM NaCl，10%（v/v）甘油，10 mM咪唑，pH7 .2 ）以0 .5-1ml/min的流速冲洗管路。将含有TSPase的粗酶液上样，带有His标签的蛋白能与Ni柱中的硫酸镍结合，将蛋白截留。上样完成后利用缓冲液B（50 mM磷酸钠缓冲，300 mM NaCl，10%（v/v）甘油，250 mM咪唑，pH7 .2）以1 ml/min的流速进行梯度洗脱，缓冲液B中的咪唑也可以与Ni柱中的硫酸镍结合，从而与带有His标签的蛋白形成竞争关系。收集穿透峰和目标峰。洗脱结束后分别用纯水和20%乙醇清洗Ni柱。洗脱液用超滤离心管4℃、6000 rpm离心10 min，脱盐除咪唑，得到纯酶液。超滤离心管的截留分子量为30 KDa。蛋白电泳结果显示在100 KDa处有一条与理论分子量一致的条带(图2)。
实施例6：温度对α-葡萄糖苷酶突变体的影响
将20 μg经实施例5纯化的重组α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L加至100 μl反应体系中（25 mM麦芽糖，100 mM磷酸钠缓冲，pH 4 .0），在不同的温度（30℃、37℃、45℃、55℃、65℃、75℃）条件下反应10 min，反应液经95℃水浴加热7 min终止反应，利用葡萄糖氧化酶法检测样品中生成的葡萄糖的量，每个反应做三组平行样。取酶活最高的一组作为相对酶活100%。
将20 μg经实施例5纯化的α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L，在不同的温度（30℃、37℃、45℃、55℃、65℃、75℃）条件下孵育一段时间（15，30，45，60 min）后，加至100 μl反应体系中（25 mM麦芽糖，100 mM磷酸钠缓冲，pH 4 .0）于37℃反应10 min，反应液经95℃水浴加热7 min终止反应，利用葡萄糖氧化酶法检测样品中生成的葡萄糖的量，每个反应做三组平行样。取酶活最高的一组作为相对酶活100%。
结果表明温度对不同α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L的影响基本一致，均为65℃条件下酶活力最高，但在60℃温育15 min后，仅保持其初始活性的43.1%。所述α-葡萄糖苷酶突变体在温度范围为30℃-55℃条件下较为稳定，温育60min后均仍保持其初始活性的80%以上。该α-葡萄糖苷酶突变体的最适反应温度范围为30℃-55℃。
实施例7：pH对α-葡萄糖苷酶突变体的影响
将20 μg经实施例5纯化的重组α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L，加至不同pH（3.0-7.0）的100 μl反应体系中（25 mM麦芽糖，100 mM磷酸钠缓冲）37℃反应10 min，反应液经95℃水浴加热7 min终止反应，利用葡萄糖氧化酶法检测样品中生成的葡萄糖的量，每个反应做三组平行样。取酶活最高的一组作为相对酶活100%，
将20 μg经实施例5纯化的重组α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L，加至不同pH（3.0-7.0）的缓冲溶液中置于4℃保存24h或48h，后加入25 mM麦芽糖（100 μl反应体系）37℃反应10 min，反应液经95℃水浴加热7 min终止反应，利用葡萄糖氧化酶法检测样品中生成的葡萄糖的量，每个反应做三组平行样。取酶活最高的一组作为相对酶活100%。
结果表明pH对不同α-葡萄糖苷酶突变体Y270F、W373L、M411F、R491K和W504L的影响基本一致，均在pH 3.0-5.0的条件范围内催化效率较高，最适反应pH为4 .0。
实施例8：最适温度及pH条件下，α-葡萄糖苷酶及其突变体比活力比较
在pH 4.0，37℃反应条件下，测定α-葡萄糖苷酶及其突变体Y270F、W373L、M411F、R491K和W504L的酶活力。突变体的酶活力与野生酶相比有明显提高，Y270F、W373L、M411F、R491K、W504L的比活力分别是野生酶的10.0倍、12.4倍、7.3倍、5.3倍和1.5倍。
表1α-糖苷酶突变体的比活力
| 酶 | wildtype | Y270F | W373L | M411F | R491K | W504L |
| 比活力/U·mg<sup>-1</sup> | 0.87 | 8.73 | 10.82 | 6.35 | 4.58 | 1.34 |
实施例9：pH对利用α-葡萄糖苷酶催化合成AA-2G的影响
催化反应采用α-葡萄糖苷酶及其突变体Y270F、W373L、M411F、R491K和W504L的粗酶液进行。200 μl反应体系中包含267 mM麦芽糖，178 mM L-AA，13 mM硫脲，pH 4.0或pH5.0的100 mM 乙酸缓冲，加入3U/g粗酶液，在37℃避光条件下反应6h，每小时取样，加入800 μl1.06%偏磷酸终止反应。产物用HPLC进行检测。
相同反应时间条件下，pH 4.0条件下生成的AA-2G的产量高于pH 5.0条件下的产量。
实施例10：反应温度对利用α-葡萄糖苷酶催化合成AA-2G的影响
催化反应采用α-葡萄糖苷酶及其突变体Y270F、W373L、M411F、R491K和W504L进行。200μl反应体系中包含267 mM麦芽糖，178 mM L-AA，13 mM硫脲，pH 4.0的100 mM 乙酸缓冲，加入3U/g粗酶液，在37℃或50℃避光条件下反应6h，每小时取样，加入800 μl 1.06%偏磷酸终止反应。产物用HPLC进行检测。
反应2h，50℃条件下生成的AA-2G的产量高于37℃条件下的产量，但随着反应时间的延长，产物AA-2G也会被降解；6h反应时间内，37℃条件下得到的AA-2G的总量更多，因而选择37℃作为较佳的反应温度。
实施例11：最适反应温度及pH条件下，利用α-葡萄糖苷酶及其突变体合成AA-2G的比较
催化反应采用α-葡萄糖苷酶及其突变体Y270F、W373L、M411F、R491K和W504L进行。200μl反应体系中包含267 mM麦芽糖，178 mM L-AA，13 mM硫脲，pH 4.0的100 mM 乙酸缓冲，加入3U/g粗酶液，在37℃避光条件下反应6h，加入800 μl 1.06%偏磷酸终止反应。产物用HPLC进行检测。
以突变体W373L作为催化剂，生成AA-2G的量达到最高为18.9 g/L，是野生酶产量的15.75倍；利用突变体Y270F、M411F、R491K和W504L作为催化剂，其产量分别是野生酶的12.75倍、12.4倍、6.8倍和4.75倍。
表2 AA-2G生成量比较
| 酶 | wildtype | Y270F | W373L | M411F | R491K | W504L |
| AA-2G（g/L） | 1.2 | 15.3 | 18.9 | 14.9 | 8.2 | 5.7 |
实施例12：不同糖基供体对催化合成AA-2G的影响
催化反应采用α-糖苷酶突变体W373L的粗酶液进行。200 μl反应体系中分别包括100g/L的麦芽糖、100g/L的麦芽低聚糖、100g/L的异麦芽糖、100g/L的蔗糖、100g/L的曲二糖和20g/L淀粉，178 mM L-AA，13 mM硫脲，pH 4.0的100 mM 乙酸缓冲，加入3U/g粗酶液，在37℃避光条件下反应6h，每小时取样，加入800 μl 1.06%偏磷酸终止反应。产物用HPLC进行检测。
反应体系中以麦芽糖作为糖基供体，催化生成AA-2G的产量最高可达18.9 g/L，是分别以麦芽低聚糖、异麦芽糖、蔗糖、曲二糖和淀粉为糖基供体生成AA-2G产量的3.5倍、5.7倍、2.2倍、4.1倍和8.6倍。
虽然本发明已以较佳实施例公开如上，但并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 南京工业大学
<120> 一种α-糖苷酶基因突变体及在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用
<141> 2020-12-02
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 852
<212> PRT
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Gly Tyr Asn Val Ala Ser Val Ala Gly Ser Lys Asn Arg Leu Arg Ala
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Arg Leu Glu Leu Ala Gly Gly Gly Gly Gly Ala Ala Pro Glu Leu Gly
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Pro Asp Val Arg Arg Leu Ser Leu Thr Ala Ser Leu Glu Thr Asp Ser
35 40 45
Arg Leu His Val Arg Ile Thr Asp Ala Asp His Pro Arg Trp Glu Val
50 55 60
Pro Gln Asp Val Ile Pro Arg Pro Ser Pro Asp Ser Phe Leu Ala Ala
65 70 75 80
Thr Arg Pro Gly Gly Gly Arg Val Leu Ser Thr Ala Thr Ser Asp Leu
85 90 95
Thr Phe Ala Ile His Thr Ser Pro Phe Arg Phe Thr Val Thr Arg Arg
100 105 110
Ser Thr Gly Asp Val Leu Phe Asp Thr Thr Pro Asn Leu Val Phe Lys
115 120 125
Asp Arg Tyr Leu Glu Leu Thr Ser Ser Leu Pro Pro Pro Gly Arg Ala
130 135 140
Ser Leu Tyr Gly Leu Gly Glu Gln Thr Lys Arg Thr Phe Arg Leu Gln
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Arg Asn Asp Thr Phe Thr Leu Trp Asn Ser Asp Ile Ala Ala Gly Asn
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Val Asp Leu Asn Leu Tyr Gly Ser His Pro Phe Tyr Met Asp Val Arg
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Ser Gly Gly Gly Gly Gly Gly Gly Ala Ala His Gly Val Leu Leu Leu
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Asn Ser Asn Gly Met Asp Val Ile Tyr Gly Gly Ser Tyr Val Thr Tyr
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Lys Val Ile Gly Gly Val Leu Asp Phe Tyr Phe Phe Ala Gly Pro Ser
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Pro Leu Ala Val Val Asp Gln Tyr Thr Gln Leu Ile Gly Arg Pro Ala
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Pro Met Pro Tyr Trp Ser Phe Gly Phe His Gln Cys Arg Tyr Gly Tyr
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Lys Asn Val Ala Asp Leu Glu Gly Val Val Ala Gly Tyr Ala Lys Ala
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Arg Ile Pro Leu Glu Val Met Trp Thr Asp Ile Asp Tyr Met Asp Ala
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Tyr Lys Asp Phe Thr Leu Asp Pro Val Asn Phe Pro Ala Asp Arg Met
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Arg Pro Phe Val Asp Arg Leu His Arg Asn Gly Gln Lys Phe Val Val
325 330 335
Ile Ile Asp Pro Gly Ile Asn Val Asn Thr Thr Tyr Gly Thr Phe Val
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Arg Gly Met Lys Gln Asp Ile Phe Leu Lys Trp Asn Gly Ser Asn Tyr
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Leu Gly Val Val Trp Pro Gly Asn Val Tyr Phe Pro Asp Phe Leu Asn
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Pro Arg Ala Ala Glu Phe Trp Ala Arg Glu Ile Ala Ala Phe Arg Arg
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Thr Leu Pro Val Asp Gly Leu Trp Val Asp Met Asn Glu Ile Ser Asn
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Phe Val Asp Pro Pro Pro Leu Asn Ala Ile Asp Asp Pro Pro Tyr Arg
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Ile Asn Asn Ser Gly Val Arg Arg Pro Ile Asn Asn Lys Thr Val Pro
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Ala Ser Ala Val His Tyr Gly Gly Val Ala Glu Tyr Asp Ala His Asn
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Leu Phe Gly Phe Leu Glu Ala Arg Ala Thr His Asp Ala Leu Leu Arg
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Asp Thr Gly Arg Arg Pro Phe Val Leu Ser Arg Ser Thr Phe Val Gly
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Ser Gly Arg Tyr Thr Ala His Trp Thr Gly Asp Asn Ala Ala Thr Trp
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Glu Asp Leu His Tyr Ser Ile Asn Thr Met Leu Ser Phe Gly Leu Phe
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Gly Ile Pro Met Ile Gly Ala Asp Ile Cys Gly Phe Gly Gly Asn Thr
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Thr Glu Glu Leu Cys Ser Arg Trp Ile Gln Leu Gly Ala Phe Tyr Pro
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Phe Ser Arg Asp His Ser Ala Ile Gly Thr Val Arg Arg Glu Leu Tyr
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Leu Trp Glu Ser Val Ala Arg Ser Ala Arg Lys Ala Leu Gly Leu Arg
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Tyr Arg Leu Leu Pro Tyr Leu Tyr Thr Leu Met Tyr Glu Ala His Thr
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Thr Gly Ala Pro Ile Ala Arg Pro Leu Phe Phe Ser Tyr Pro Gly Asp
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Val Glu Thr Tyr Gly Ile Asp Arg Gln Phe Leu Leu Gly Arg Gly Val
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Phe Pro Ala Gly Arg Trp Phe Ser Leu Tyr Asp Phe Ser Leu Ala Val
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Val Asn Val His Val Ala Gly Gly Asn Ile Leu Thr Leu Gln Gln Pro
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Ser His Val Val His Asp Ser Tyr Ala Pro Ser Arg Thr Met Ala Ile
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Phe Ala Val Tyr Ala Asn Gly Val Gln Val Asn Ala Ser Thr Ala Val
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Gly Gly Ala Ala Gly Ser Pro Glu Lys Gly Ala Leu Gly Val Ala His
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<210> 2
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagatac ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat atgaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct agaagcactt tcgttggttc tggtagatat 1500
actgctcatt ggactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 3
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagattc ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat atgaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct agaagcactt tcgttggttc tggtagatat 1500
actgctcatt ggactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 4
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagatac ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat atgaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct agaagcactt tcgttggttc tggtagatat 1500
actgctcatt ggactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 5
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagatac ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat ttcaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct agaagcactt tcgttggttc tggtagatat 1500
actgctcatt ggactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 6
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagatac ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat atgaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct aaaagcactt tcgttggttc tggtagatat 1500
actgctcatt ggactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 7
<211> 2559
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
ggatataatg tcgcatcagt cgcaggaagt aaaaacagat tgagagcaag attggagttg 60
gcaggaggag gtggtggtgc tgctccagaa ttgggtccag atgttagaag attgtctttg 120
actgcttctt tggagactga ttctagattg cacgttagaa ttactgatgc tgatcatcct 180
agatgggaag ttccacaaga tgttattcca agaccttctc cagattcttt cttggctgct 240
actagaccag gtggtggtag agttttgtct actgctactt ctgatttgac ttttgctatt 300
cacacttctc cattcagatt cactgttact agaagaagca ctggtgacgt tttgtttgat 360
actactccta acttggtttt caaggataga tacttggagt tgacttcttc tttgcctcca 420
cctggtagag cttctttgta tggtttgggt gaacaaacta agagaacttt cagattgcaa 480
agaaacgata ctttcacttt gtggaactct gatattgctg ctggtaacgt tgatttgaac 540
ttgtacggtt ctcatccatt ttacatggat gttagaagcg gtggtggtgg tggtggtggt 600
gctgctcatg gtgttttgtt gttgaactct aatggtatgg atgttattta tggtggttct 660
tatgttactt acaaggttat tggtggtgtt ttggatttct actttttcgc tggtccttct 720
cctttggctg ttgttgatca atacactcaa ttgattggta gaccagctcc tatgccttac 780
tggtctttcg gttttcacca atgtagatac ggttataaga acgttgctga tttggaaggt 840
gttgttgctg gttacgctaa agctagaata ccattggagg ttatgtggac tgatattgat 900
tacatggatg cttataagga tttcactttg gatccagtta acttccctgc tgatagaatg 960
agacctttcg ttgatagatt gcacagaaac ggtcaaaagt tcgttgttat tattgatcct 1020
ggtattaatg ttaacactac ttatggtact ttcgttagag gtatgaaaca agatattttc 1080
ttgaagtgga atggttctaa ctacttgggt gttgtttggc caggtaacgt ttacttccca 1140
gatttcttga acccaagagc tgctgaattt tgggctagag agattgctgc tttcagaaga 1200
actttgccag ttgatggttt gtgggttgat atgaacgaga tttctaactt tgttgatcca 1260
cctccattga acgctattga tgatcctcca tatagaatta ataactctgg tgttagaaga 1320
ccaattaaca acaagactgt tccagcttct gctgttcatt acggtggtgt tgctgagtac 1380
gatgctcata atttgttcgg tttcttggaa gctagagcta ctcacgatgc tttgttgaga 1440
gatactggta gaagaccatt cgttttgtct agaagcactt tcgttggttc tggtagatat 1500
actgctcatt tgactggtga caacgctgct acttgggaag atttgcatta ctctattaac 1560
actatgttgt ctttcggttt gttcggtatt ccaatgattg gtgctgatat ttgtggtttc 1620
ggtggtaaca ctactgagga attgtgttct agatggattc aattgggtgc tttctaccca 1680
ttttctagag atcactctgc tattggtact gttagaagag aattgtactt gtgggagtct 1740
gttgctagaa gcgctagaaa ggctttgggt ttgagataca gattgttgcc atatttgtac 1800
actttgatgt atgaagctca cactactggt gctcctattg ctagaccttt gtttttctct 1860
tacccaggtg acgttgagac ttatggtatt gatagacaat tcttgttggg tagaggtgtt 1920
ttggtttctc cagttttgga gcctggtgct actactgtta ctgcttactt ccctgctggt 1980
agatggttct ctttgtacga tttttctttg gctgttgcta ctaagactgg taaaagagtt 2040
actttgccag ctcctgctga tactgttaac gttcatgttg ctggtggtaa tattttgact 2100
ttgcaacaac cagctttgac ttcttctaga gttagacaat ctgttgttca cttgttggtt 2160
gctttggctg atgatggtac tgctactggt gacttgttct tggatgatgg tgagtctcca 2220
gaaatggctg gtcctagaag cagatggtct caaattaagt tctctggtgc tactgaatct 2280
ggtggtggtg ttgttagagt tagaagccac gttgttcatg attcttacgc tccttctaga 2340
actatggcta ttggtaaagt tgttttgatg ggtttgagaa gcgctgctcc acctaagggt 2400
ttcgctgttt acgctaacgg tgttcaagtt aacgcttcta ctgctgttgg tggtgctgct 2460
ggttctccag aaaagggtgc tttgggtgtt gctcacgtct caggtttgac acttgttgtt 2520
ggacaggagt ttgatttgaa ggttgttatg acttattaa 2559
<210> 8
<211> 31
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
tgtagattcg gttataagaa cgttgctgat t 31
<210> 9
<211> 30
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
ataaccgaat ctacattggt gaaaaccgaa 30
<210> 10
<211> 30
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
tgttgtttgg ccaggtaacg tttacttccc 30
<210> 11
<211> 31
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
acctggccaa acaacaccca agtagttaga a 31
<210> 12
<211> 31
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
tgggttgatt tcaacgagat ttctaacttt g 31
<210> 13
<211> 30
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
ctcgttgaaa tcaacccaca aaccatcaac 30
<210> 14
<211> 30
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
tttgtctaaa agcactttcg ttggttctgg 30
<210> 15
<211> 31
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 15
aagtgctttt agacaaaacg aatggtcttc t 31
<210> 16
<211> 30
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 16
gctcatttga ctggtgacaa cgctgctact 30
<210> 17
<211> 33
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 17
accagtcaaa tgagcagtat atctaccaga acc 33
Claims (10)
1.一种α-葡萄糖苷酶突变体，其特征在于，所述突变体是在氨基酸序列如SEQ NO.1所示的α-葡萄糖苷酶发生了一个或多个氨基酸位点的突变；所述突变经过以下至少一种突变情况所得：
将第270位酪氨酸突变为苯丙氨酸，简称Y270F；
或/和将第373位色氨酸分别突变为亮氨酸，简称W373L；
或/和将第411位甲硫氨酸突变为苯丙氨酸，简称M411F；
或/和将第491位精氨酸突变为赖氨酸，简称R491K；
或/和将第504位色氨酸突变为亮氨酸，简称W504L。
2.编码权利要求1所述的α-葡萄糖苷酶突变体的多核苷酸序列。
3.一种重组载体，携带编码权利要求3所述的多核苷酸序列。
4.一种重组细胞，可表达权利要求2所述的α-葡萄糖苷酶突变体。
5.一种生产α-葡萄糖苷酶突变体的方法，其特征在于，由权利要求5所述重组细胞发酵培养获得。
6.权利要求1所述α-葡萄糖苷酶突变体在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用。
7.α-葡萄糖苷酶突变体在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用，其特征在于，由选自由以下组成中的一种及以上：权利要求3所述的α-葡萄糖苷酶突变体、含有权利要求4所述的重组载体的重组细胞、权利要求5所述的重组细胞的培养物和权利要求5所述的重组细胞培养物上清作为催化剂，以包含但不限于麦芽糖、麦芽低聚糖、异麦芽糖、蔗糖、曲二糖和淀粉为糖基供体、L-抗坏血酸为糖基受体，转化生成2-O-α-D-葡萄糖基-L-抗坏血酸。
8.根据权利要求7所述的应用，其特征在于：所述糖基供体包含但不限于100g/L的麦芽糖、100g/L的麦芽低聚糖、100g/L的异麦芽糖、100g/L的蔗糖、100g/L的曲二糖和20g/L淀粉。
9.根据权利要求7所述的应用，其特征在于，所述糖基受体为30-50g/L L-抗坏血酸，催化剂浓度为10-20 U/mL。
10.根据权利要求7所述的应用，其特征在于，所述反应在30℃-55℃、pH4.0-5.0条件下反应1-6h。
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